Human parainfluenza viruses (HPIV1, 2, and 3) are the second leading cause of pediatric hospitalization in the United States due to respiratory viral infection. In the immunocompromised, these pathogens also cause prolonged illness and often death. Our long-term goal is to understand how parainfluenza viruses cause disease and induce immunity so that specific antiviral drugs and vaccines, which are currently unavailable, can be developed. The parainfluenza virus fusion (F) envelope glycoprotein is found on the surfaces of virions and infected cells. We hypothesize that the parainfluenza virus F protein regulates viral infection and the host response by its multiple functional activities of promoting membrane fusion and immunogenicity. Recent determinations of high-resolution structures of parainfluenza virus F proteins in both prefusion (native) and postfusion (hairpin) forms now provides a structural basis to investigate molecular mechanisms by which the F protein regulates the biology of parainfluenza viruses. The objective of this application is to understand how structural changes by parainfluenza virus F proteins are regulated (Specific Aim 1) and how they help determine pathogenicity (Specific Aim 2) and immunogenicity (Specific Aim 3). In Specific Aim 1, we will determine how HPIV3 F protein structural changes are regulated during membrane fusion. We will test the hypothesis that F protein refolding and membrane fusion are regulated by residues in regions that undergo dramatic structural changes between prefusion and postfusion F protein structures. Mutational analyses on heptad repeat (HR) regions in the HPIV3 F protein will be performed. Understanding how the F protein structure is stabilized and then triggered to refold during membrane fusion will provide a greater understanding of protein-mediated membrane fusion, a fundamental mechanism common to all enveloped viruses. In Specific Aim 2, we will investigate how F protein fusogenicity causes Sendai virus pathogenicity in mice. We will determine mechanisms by which a hyperfusogenic recombinant Sendai virus variant rSeV-F-L179V and other virus variants induce greater pathogenicity in mice. Studying Sendai virus pathogenesis in its natural host, the mouse, will provide an understanding of the role of the F protein in pathogenesis. These studies will also support efforts to treat human parainfluenza virus infection with novel therapeutics. In Specific Aim 3, we will increase the immunogenicity of a Sendai virus vaccine that expresses the HPIV3 F protein. We will determine how modifications of the Sendai virus vector and the expressed structural form of the HPIV3 F protein antigen help determine immunogenicity and potential immunopathology. Because the F protein is conserved among all of the paramyxoviruses, an understanding of how the HPIV3 F protein determines immunity and immunopathology may assist in the development of vaccines for other important respiratory paramyxoviruses like the other human parainfluenza viruses (HPIV1, HPIV2, and HPIV4) and respiratory syncytial virus (RSV). PUBLIC HEALTH RELEVANCE: Human parainfluenza viruses are a leading cause of hospitalization of U.S. children, and they can be deadly in immunocompromised patients. Our proposed work will help understand how parainfluenza viruses cause disease at the molecular level. These studies will help efforts to develop new drugs and vaccines against parainfluenza viruses.